The actin-based cytoskeleton of the intestinal epithelial cell (enterocyte) plays critical roles in the absorptive and barrier functions of the intestinal mucosa. The apical brush border (BB) domain of the enterocyte is a complex, highly ordered array of actin filaments and associated actin-binding proteins including numerous members of the myosin family of actin-based molecular motors, a number of which are mutated in inherited human diseases. The proposed studies will test a number of inter-related hypotheses regarding the functions for myosins in the enterocyte. Because the enterocyte represents one of the best model systems for investigating the actin cytoskeleton, these studies will provide insights of both specific and general significance. The initial focus will be on myosin-la (Myola) and two other myosins with which it has functional synergy, myosin-VI (Myo6) and myosin-le (Myole). Myola forms links between the microvillar (MV) membrane and underlying actin core. Myo6 is associated with the clathrin-rich interMV domain, while Myole is found on both apical and basolateral membranes. Assessment of function is facilitated by availability of mutant mouse lines for these 3 myosins. There are marked compositional and structural defects in the BB membrane and underlying cytoskeleton in enterocytes of mice lacking Myola including the loss of Myo6 and Myole. Hypotheses will be tested to examine the role of these myosins in a number of critical cellular processes. These include the maintenance BB membrane composition and structural integrity, regulation of BB actin assembly dynamic and endocytosis. Hypotheses investigating the role of these myosins in the barrier functions of the enterocyte will address the role of these myosins in the regulation of the paracellular barrier and response to mucosal injury. In addition to in in vivo and in vitro studies using the mutant mouse lines, parallel studies will be performed using an intestinal epithelial cell line in which Myola, Myole or Myo6 functions in regulating actin assembly, endocytosis, and wound healing have been disrupted through expression of GFP-tagged, dominant negative-tails of these myosins. These studies should provide critical insights into the role of myosins in enterocyte function-information essential to understand human diseases of the intestine including inflammatory bowel disease and malabsorption syndromes. Moreover, many myosins are targets for human diseases, including 2 of the 3 to be investigated.